The invention relates to a method for centrifugal particle separation.
Research in the area of organ functions on the cellular level has enormously expanded during the past years. Basic physiological research today centers on describing specific functions of the differentiated and specialized cell types which form the individual tissue types and which, in combination, are ultimately responsible for performing the central tasks of the body's organs.
The primary precondition for the further development of this important direction in research is the availability of ever more efficient cell separation methods. Today, immunological separation techniques seem to be very promising for this purpose. They are always based on the expression of typical cellular antigens which are identified by means of highly specific antibodies and ultimately used for separation. Anchoring the antibodies to magnetic particles, for example, can cause the cells, through these proteins, to be bound to the particles as well. In the ideal case, this process can be used in an attractively simple manner to separate the bound cells by means of a magnet.
However, cell-specific antibodies are often extremely species-specific (and therefore frequently unavailable) and furthermore very costly. In addition to these limitations, which in practice are often decisive, the analizability of antigen structures for successful cell separation quickly meets insurmountable obstacles whenever it is to be used for separating cell types that are initially tightly bonded together within tissue types--in contrast to blood cells, for instance, which are present in a physiological suspension.
Thus, the first and foremost prerequisite is the complete dissociation and suspension of such cells from their union with the native tissue. This can be achieved only by the action of complex proteolytic mixtures which are apt in their attack to change substantially and unavoidably the antigen pattern of the tissue cells. Antigens which are frequently detached or masked or even newly developed or expressed in a non-specific manner during proteolysis soon make the subsequent immunological separation technique inefficient. Numerous foreign cells will typically creep into the final suspension of the "purified" target cell type. As a result, there is currently a surge of false announcements in the technical literature.
Certain physical or physical-chemical cell characteristics survive the action of proteolytic enzymes substantially more reliably than immunologically identifiable cell properties. This includes on the one hand size, form and aggregability of the cells and on the other hand their specific weight which, under given physiological conditions, substantially depends on the ion and water permeability of the cell membranes or the osmotic pressure present within the cells. Each of these physical or physical-chemical quantities can be used as a separation parameter for a successful cell separation if the cells are exposed to the gravitational field of a suitable centrifuge. Customarily, cell separation in centrifuge vessels takes place in liquid media of a certain density which are layered as so-called "discontinuous or continuous density gradients." The first task of these media is to stabilize the intended cell separation against thermal convection and mechanical vibration. The sedimentation rate v depends on the interrelationship expressed by the following formula: ##EQU1## where d is the cell radius, .delta..sub.Z and .delta..sub.M the specific density of the cells and the medium, respectively, .mu. the viscosity of the separation medium, .omega. the angular velocity and r the rotor radius.
On this basis, the following two techniques, which in principle can be selected at will but cannot be combined with complete consistency, are currently practiced in centrifugation processes:
1. "Zonal Centrifugation"
Here, the cells are separated in a gradient of the selected separation medium which becomes increasingly dense in sedimentation direction but is nevertheless relatively shallow and continuous or formed in steps (various products are available on the market, for example, Ficoll, Metrizamide, Percoll, etc.), such that none of the cell types can find an isopycnic density range (one which corresponds to its own specific density). As a result, all cell types would collect again on the bottom of the separation vessel if centrifugation were not interrupted at the appropriate time. Separation occurs primarily based on the different size of the cell types (see above formula).
2. "Isopycnic Centrifugation"
In this case, a density gradient is introduced which also includes ranges of the same specific density as that of the cells. If a cell type reaches the gradient range which is "isopycnic" to it, its sedimentation rate approaches zero (see above formula) and cells of different specific weights then separate within the gradient, provided the gradient profile in the centrifuge vessel has a suitable spatial characteristic. Depending on the separation task, it is better to load linear or convex or concave gradients.
DE-OS 34 04 236 discloses the design of a rotor which is suitable for such cell separation, permitting the use of the aforementioned centrifugation methods in that the interior of the separation vessel remains accessible during the entire centrifugation period and that the gradient can be aspirated via a corresponding cannula. An additional advantage is that the entire rotor can be autoclaved, thus providing the conditions for a sterile (aseptic) process and, possibly, a subsequent long-term cultivation of the separated cells in the tissue laboratory.
Many years of experience with this rotor have shown design characteristics that are well worth preserving but have also revealed the following design problems and limitations:
a) Coriolis forces exist within the centrifuge vessel as shown in FIG. 1. A particle within the rotating (arrow 4) centrifuge vessel 1 would move along intended line 2 if said Coriolis force is not taken into account. In effect, however, this force acts on the particle such as to cause it to move along line 2. As a result, cell bands 6, 8 separated in gradient 7 are shaped or deformed as shown in FIG. 2. Fractionating these cell bands, 6, 8 via the tip of a cannula 5 terminating at the end of centrifuge vessel 1 causes partial smudging of the cell separation. The full separation capacity of the unit is therefore ultimately not usable because parts of band 6 continue to be eluted when band 8 has already arrived at the tip. PA1 b) The cannula arrangement permits fractionation of the separated bands only by means of suction. While the centrifuge is running, the required suction must exceed the centrifugal force. Since this force must be as high as possible to prevent vortexing of the separated cells, the vacuum required for sucking off the cells must be so considerable that it may cause partial "degassing" of physically dissolved physiological gases (oxygen, carbon dioxide, nitrogen) in the cell's interior which can be associated with cell damage. Furthermore, for practical reasons it is rarely possible to achieve continuous elution. The use of peristaltic pumps for the continuous removal of cells would in any case be deleterious to almost all cell types. In addition, there is the constant danger of vortexing if, during the removal from the cannula entry of the syringes that are frequently used for aspirating the gradient, the volume remaining in the cannula is thrown back into the tip of the centrifuge glass.